Stem cells represent unique cell populations that have the ability to undergo both self-renewal and differentiation. Although "adult" stem cells may develop into only a limited number of cell types, in contrast, early repopulating stem cells have the ability to initiate lineage-specific differentiation programs and to form numerous cell types in vitro. Megakaryocytopoiesis involves the commitment of hematopoietic stem cells, and the proliferation, maturation, and terminal differentiation of megakaryocytic progenitors. Characterization of megakaryocyte cytokines and regulators are important not only for improving our understanding of megakaryocytopoiesis and stem cell biology, but also for potential clinical applications. However, characterization of megakaryocytopoiesis, and examination of cellular development, gene expression or protein composition within the hematopoietic microenvironment have been difficult because of the relatively low cell frequency of megakaryoctyes: megakaryocytes represent only 0.015% to 0.03% of the total number of nucleated cells in human marrow. Furthermore, analysis of the role of endogenously secreted regulators by ELISA and other conventional bioassay techniques, which detect supernatant from heterogeneous cell populations, is difficult and imprecise because endogenous proteins are sometimes secreted at very low levels. To aid the study of endogenous secreted proteins and the process of megakaryocyte differentiation, this SBIR will use single cell gel microdrop (GMD) encapsulation technology and flow cytometry to develop a multi-parameter assay to detect cell surface marker expression and cytokine secretion simultaneously on single, viable hematopoietic stem/progenitor cells. This assay will facilitate sensitive determination of secretion at the single cell level together with clear cut identification of each immunostained, functionally active megakaryocyte. PUBLIC HEALTH RELEVANCE: The proposed single cell assay will facilitate the study of endogenous secreted proteins and the process of megakaryocyte differentiation, and aid the development of stem cell therapies. [unreadable] [unreadable] [unreadable]